The purpose of this shared equipment grant if to obtain instrumentation to upgrade our capability for monitoring fluorescent molecules in living cells. Our objective is to extend well-developed fluorescence techniques, for example, fluorescence quantitation, resonance energy transfer, photobleaching and spectrofluorometry, to the light microscopy. The intention, with each of these analytical procedures, is to preserve spatial information in a digital format and to apply a range of digital image processing and three-dimensional reconstruction methods as an essential element in the design and interpretation of experiments. The proposals reflect diverse interests, but share a need for this capability. In each proposal, we have developed the scientific problem, then indicate how this instrumentation will play a dey role in its solution. In most instances, the information being sought simply cannot be obtained in any other way. Three examples are (a) the changes in the three-dimensional structure of the microtubule network of a human PMN responding to a chemotactic signal, (b) the effect of an introduced actin crosslinking protein on the shape of a living cell, or (c) the dynamics of the uptake and degradation of lipoproteins by living cells. The instrumentation will allow two conceptually distinct extensions of our basic approach. The computing capability will allow us to apply the full range of available image processing and three-dimensional reconstruction methods to a wide range of biological problems. Each of the proposals makes wide use of this capability to analyze fully the distribution of molecules or, in one instance, to develop morphometric methods to analyze atherosclerotic lesions. The analytic capability will allow us to do sophisticated fluorescence experiments. An example is the measurement, by resonance energy transfer, of the interaction of two macromolecules in living cells with retention of the spatial information. The computing and analytic capabilities are integrated completely. We expect, for example, to be able to collect, store, and analyze multiple image frames after a photo-bleaching pulse, then use this information to determine, by examining the spatial redistribution of fluorescence as a function of time, the kinetic parameters for exchange of molecules into biological structures. This approach represents a novel, fundamentally important extension of methods to analyze cellular problems.